Raf protein kinase therapeutics

ABSTRACT

It is a general object of this invention to provide a DNA segment comprising a Raf gene in an antisense orientation downstream of a promoter. It is a specific object of this invention to provide a method of inhibiting Raf expression comprising expressing an antisense Raf gene in a cell such that said Raf expression is inhibited. It is a further object of the invention to provide a method of inhibiting Raf kinase activity comprising replacement of a serine or threonine amino acid within the Raf gene for a non-phosphorylated amino acid.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates, in general, to methods of inducing a therapeutic effect. In particular, the present invention relates to therapeutic uses of Raf protein kinases.

[0003] 2. Background Information

[0004] Raf serine- and threonine-specific protein kinases are cytosolic enzymes that stimulate cell growth in a variety of cell systems (Rapp, U. R., et al. (1988) In The oncogene handbook; T. Curran, E. P. Reddy, and A. Skalka (ed.) Elsevier Science Publishers; The Netherlands, pp.213-253; Rapp, U. R., et al. (1988) Cold Spring Harbor Sym. Quant. Biol. 53:173-184; Rapp, U. R., et al. (1990) In: Curr. Top. Microbiol. Immunol. Potter and Melchers (eds), Berlin, Springer-Verlag 166:129-139). Three isozymes have been characterized: c-Raf (Raf-1) (Bonner, T. I., et al. (1986) Nucleic Acids Res. 14:1009-1015). A-Raf (Beck, T. W., et al. (1987) Nucleic Acids Res. 15:595-609), and B-Raf (Ikawa, S., et al. (1988) Mol. Cell. Biol. 8:2651-2654; Sithanandam, G. et al. (1990) Oncogene 5:1775). These enzymes differ in their expression in various tissues. Raf-1 is expressed in all organs and in all cell lines that have been examined, and A- and B-Raf are expressed in urogenital and brain tissues, respectively (Storm, S. M. (1990) Oncogene 5:345-351).

[0005] Raf genes are proto-oncogenes: they can initiate malignant transforation of cells when expressed in specifically altered forms. Genetic changes that lead to oncogenic activation generate a constitutively active protein kinase by removal or interference with an N-terminal negative regulatory domain of the protein (Heidecker, G., et al. (1990) Mol. Cell. Biol. 10:2503-2512; Rapp, U. R., et al. (1987) In Oncogenes and cancer S. A. Aaronson, J. Bishop, T. Sugimura, M. Terada, K. Toyoshima, and P. K. Vogt (ed.) Japan Scientific Press, Tokyo). Microinjection into NIH 3T3 cells of oncogenically activated but not wild-type versions of the Raf-protein prepared with Escherichia coli expression vectors results in morphological transformation and stimulates DNA synthesis (Rapp, U. R., et al. (1987) In Oncogenes and cancer; S. A. Aaronson, J. Bishop, T. Sugimura, M. Terada, K. Toyoshima, and P. K. Vogt (ed.) Japan Scientific Press, Tokyo; Smith, M. R., et al. (1990) Mol. Cell. Biol. 10:3828-3833). Thus, activated Raf-1 is an intracellular activator of cell growth. Raf-1 protein serine kinase is a candidate downstream effector of mitogen signal transduction, since Raf oncogenes overcome growth arrest resulting from a block of cellular ras activity due either to a cellular mutation (ras revertant cells) or microinjection of anti-ras antibodies (Rapp, U. R., et al. (1988) In The Oncogene Handbook, T. Curran, E. P. Reddy, and A. Skalka (ed.), Elsevier Science Publishers; The Netherlands, pp.213-253; Smith, M. R., et al. (1986) Nature (London) 320:540-543).

[0006] c-Ras function is required for transformation by a variety of membrane-bound oncogenes and for growth stimulation by mitogens contained in serum (Smith, M. R., et al. (1986) Nature (London) 320:540-543). Raf-1 protein serine kinase activity is regulated by mitogens via phosphorylation (Morrison, D. K., et al. (1989) Cell 58:648-657), which also effects subcellular distribution (Olah, Z., et al. (1991) Exp. Brain Res.84:403; Rapp, U. R., et al. (1988) Cold Spring Harbor Sym. Quant. Biol. 53:173-184).

[0007] Raf-1 activating growth factors include platelet-derived growth factor (PDGF) (Morrison, D. K., et al. (1988) Proc. Natl. Acad. Sci. USA 85:8855-8859), colony-stimulating factor 1(Baccarini, M., et al. (1990) EMBO J. 9:3649-3657), insulin (Blackshear, P. J., et al. (1990) J. Biol. Chem. 265:12131-12134; Kovacina, K. S., et al. (1990) J. Biol. Chem. 265:12115-12118), epidermal growth factor (EGF) (Morrison, D. K., et al. (1988) Proc. Natl. Acad. Sci. USA 85:8855-8859), interleukin 2 (Turner, B. C. et al. (1991) Proc. Natl. Acad. Sci. USA 88:1227), and interleukin 3 and granulocyte-macrophage colony-stimulating factor (Carroll, M. P., et al (1990) J. Biol. Chem. 265:19812-19817). Upon mitogen treatment of cells, the transiently activated Raf-1 protein serine kinase translocates to the perinuclear area and the nucleus (Olah, Z., et al. (1991) Exp. Brain Res. 84:403; Rapp, U. R., et al. (1988) Cold Spring Harbor Sym. Quant. Biol. 53:173-184). Cells containing activated Raf are altered in their pattern of gene expression (Heidecker, G., et al. (1989) In Genes and signal transduction in multistage carcinogenesis, N. Colburn (ed.), Marcel Dekker, Inc., New York. pp. 339-374), and Raf oncogenes activate transcription from Ap-1/PEA3-dependent promoters in transient transfection assays (Jamal, S., et al. (1990) Science 344:463-466; Kaibuchi, K., et al. (1989) J. Biol. Chem. 264:20855-20858; Wasylyk, C., et al. (1989) Mol. Cell. Biol. 9:2247-2250).

[0008] There are at least two independent pathways for Raf-1 activation by extracellular mitogens: one involving protein kinase C (KC) and a second initiated by protein tyrosine kinases (Blackshear, P. J. , et al. (1990) J. Biol. Chem. 265:12131-12134; Kovacina, K. S., et al. (1990) J. Biol. Chem. 265:12115-12118; Morrison, D. K., et al. (1988) Proc. Natl. Acad. Sci. USA 85:8855-8859; Siegel, J. N., et al. (1990) J. Biol. Chem. 265:18472-18480; Turner, B. C. et al. (1991) Proc. Natl. Acad. Sci. USA 88:1227). In either case, activation involves Raf-1 protein phosphorylation. Raf-1 phosphorylation may be a consequence of a kinase cascade amplified by autophosphorylation or may be caused entirely by autophosphorylation initiated by binding of a putative activating ligand to the Raf-1 regulatory domain, analogous to PKC activation by diacylglycerol (Nishizuka, Y. (1986) Science 233:305-312).

SUMMARY OF THE INVENTION

[0009] It is a general object of this invention to provide a construct comprising a DNA segment comprising a Raf gene in an antisense orientation downstream of a promoter.

[0010] It is a specific object of this invention to provide a method of inhibiting Raf expression comprising expressing an antisense Raf gene in a cell such that said Raf expression is inhibited.

[0011] It is a further object of the invention to provide a method of inhibiting Raf kinase activity comprising replacing a serine or threonine amino acid within the Raf gene for an amino acid not susceptible to phosphorylation.

[0012] Further objects and advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1. Schematic diagram of murine and human c-raf-1 cDNAs and expression plasmids used. GMA and GMS contain Stu1 restriction fragments of the mouse c-raf-1 cDNA. HCR an N-terminal HincII fragment of the human cDNA. p301 consists of all the coding sequence of a mutant human c-raf-1 cDNA. The lysine(375) to tryptophan (K→W) mutation in the ATP-binding site is indicated (Heidecker, G. et al. Molec. Cell. Biol. 10:2503-2512 (1990)). Restriction fragments were cloned in both sense and antisense orientation. NIH/3T3 cells were transfected with sense and antisense plasmids, and with the pMNC vector as control. G418-resistant (400 μg ml⁻¹) colonies containing more than 50 cells were counted after three weeks. The pMNC vector served as internal standard. The experiment was repeated three times (twice for HCR) with different batches of plasmid preparations. Variations between experiments were in the order of 10% but did not affect the ratios between the different constructs shown. CR1-3, conserved regions; ATP, ATP-binding domain; LTR, mouse Moloney virus long terminal repeat; NEO, neomycin-resistance gene; CMV, cytomegalovirus immediate early promoter.

[0014]FIG. 2. Morphological reversion of raf-transformed cells by transfection with raf antisense and mutant constructs. a. p48 raf-transformed 208/F12 (Schultz, A. M. et al. Oncogene 2:187-193 (1988)) or b. v-raf-transformed F4 (Rapp, U. R., et al. Proc. Natl. Acad. Sci. U.S.A. 80:4218-4222 (1983)) fibroblasts were transfected with plasmids p301-1 (sense) and 301-2 (antisense) or GMS-7 (sense) and GMS-8 (antisense), respectively, as well as with the pMNC vector. Monolayer growth with minor irregularities and a decreased ability to form soft agar colonies was categorized as partial reversion. Flat clones showed no areas of overgrowth and did not form colonies in soft agar. c. A representative analysis of Raf protein expression in individual cell clones. t. Transformed; im. intermediate; f. flat (clone GMS-8/2); f* (clone GMS-8/3).

[0015]FIG. 3. Mitogen responsiveness and proliferative capacity of Raf-depleted cells. a. DNA synthesis induced by serum or TPA in serum-starved cells is depicted as the number of nuclei incorporating ³H-thymidine. b. Long term growth curves GMS-7 is a pool of 10 clones transfected with sense DNA. GMS-8/2 and GMS-8/3 are flat clones reverted with antisense DNA. a,

, starved cells; □, TPA-induced cells; ▪, SERUM-induced cells. b, □, F4;

, GMS-7b; O GMS-8/2; O GM-8/3.

[0016]FIG. 4. Time course of Raf-1 mobility shift upon growth factor treatment. Cells (10⁷) expressing either wild-type (HER14) or kinase-negative (DK721A) EGF-R were stimulated at 37° C. with 40 nM EGF for the times indicated, lysed, and subjected to immunoprecipitation with anti-SP63 polyclonal antiserum. Immunoprecipitated proteins were separated by 7.5% SDS-PAGE, transferred to nitro-cellulose, and probed with the same antiserum. Immunoreactive proteins were detected with ¹²⁵I-labeled protein A, and autoradiographs were exposed for 12 h. Each lane represents immunoprecipitates from 10⁷ cells. Lanes: 2 through 5. HER14 cell; 7 and 8, DK721A cells: 9 and 10, DK721A cells with competing SP63 peptide (10 μg/ml); 1, 6, 9, and 11, marker proteins of 97 and 67 kDa.

[0017]FIG. 5. Kinase acidity upon EGF treatment HER14 and DK721A cells. Monolayer cultures of HER14 or K721A cells were incubated in the presence or absence of 40 nM EGF for 10 min at 37° C. Lysates were centrifuged, and the resulting supernatants were immunoprecipitated with Raf-1 antiserum. Immunocomplexes were assayed for kinase activity using peptide (IVQQFGFQRRASDDGKLTD) as substrate. In the absence of peptide, immune complex kinase assays with unstimulated cells yielded ≦5% of counts observed in the peptide assay with stimulated cells. No counts were incorporated when a modified version of this peptide was used, in which serine in position 12 was replaced by alanine, and position 5 retained the Raf-1-specific tyrosine.

[0018]FIG. 6. Association of Raf-1 with ligand-activated EGF-R in HER14, DK721A, or A431 cells. Density-arrested and serum-starved HER14, DK721A, or A431 cells were stimulated for 10 min with 40 nM EGF at 37° C. before lysis with RIPA buffer and immunoprecipitation. (A) Immunoprecipitates from HER14 and DK721A cells with anti-v-Raf 30K polycolonal antiserum or with a monoclonal EGF-R antibody (108) were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose. EGF-R was detected by incubating with a polyclonal antiserum (RK2) against the EGF receptor (Margolis, B., et al. (1989) Cell 57:1101-1107), followed by ¹²⁵I-labeled protein A labeling. Exposure times for immunoblots were 3 days (lanes 1 through 4) or 1 day (lanes 5 and 6). (B and C) Immunoprecipitates from EGF-treated and control A431 cells with Raf-1 specific anti-v-Raf 30K antiserum or monoclonal anti-EGF-R antibody 108 were blotted, and the blots were developed sequentially with EGF-R antiserum RK2 (B) and anti-v-Raf 30K (C). Exposure times were 3 days and 1 day for panels B and C, respectively.

[0019]FIG. 7. Phosphoamino acid analysis of the immunoprecipitated Raf-1 protein from EGF-treated and untreated HER14 cells, HER14 cells (10⁷) were phosphate starved for 16 h, labeled with 1 mCi of [³²P]phosphate for 3 h at 37° C., and treated with 40 nM EGF for 10 min at 37° C. Cells were lysed in RIPA buffer and immunoprecipitated. Proteins were separated by 7.5% SDS-PAGE, the Raf-1 bands were cut out of the gel, and the protein was electroeluted. From the electroeluted Raf-1 protein 1,960 cpm was recovered from the EGF-treated cells and 1,111 cpm was recovered from the untreated cells. The proteins were hydrolyzed for 2 h at 110° C. in 6 N hydrocloric acid. Phosphoamino acid analysis was performed at pH 1.8 as described by Cooer et al. (Cooper, A. A., et al. (1983) Methods in Enzymol. 99:387-402). The Raf-1 protein showed a shift in mobility when part of the electroeluted protein was rerun on 7.5% SDS-PAGE.

[0020]FIG. 8. Independence of EGF-medicated Raf-1 activation from PKC. HER14 cells (10⁷) were incubated for 48 h with or without 200 ng TPA and stimulated with either 100 ng of TPA for 20 min at 37° C. Calls were lysed in RIPA buffer, equal amounts of protein were immunoprecipitated with anti-v-Raf 30-kDa antiserum and electrophoresed, and the separated proteins were blotted onto nitrocellulose. The blot was incubated with the same antibody and then labeled with ¹²⁵I-labeled protein A.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to the Raf protein kinase inhibitors and methods of use thereof.

[0022] In one embodiment, the present invention relates to antisense expression constructs comprising a Raf protein kinase gene. Raf-1 function is inhibited by expressing c-raf-1 antisense RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1 interferes with proliferation of normal NIH/3T3 cells and reverts raf-transformed cells. In revertant cells, DNA replication induced by serum or TPA is eliminated or reduced proportionately to the reduction in Raf protein levels. Expression of a kinase-defective Raf-1 mutant (craf301) or a regulatory domain fragment (HCR) inhibits serum-induced NIH/3T3-cell proliferation and raf transformation even more efficiently. Inhibition by antisense RNA or craf301 blocks proliferation and transformation by Ki- and Ha-ras oncogenes. Thus, raf functions as an essential signal transducer downstream of serum growth factor receptors, protein kinase C and ras.

[0023] In another embodiment, the present invention relates to a method of inhibiting Raf expression comprising expressing an antisense Raf gene (more specifically, Raf-1) in a cell such that said Raf expression is inhibited.

[0024] In another embodiment, the present invention relates to inhibitory peptides of Raf derived from Raf kinase specific substrate sequences. Phosphorylation sites of Raf substrates can be determined which are expected to yield consensus phosphorylation site motifs for the various Raf isozymes. Studies which gave rise to the present invention demonstrate that Raf is the subject of autophosphorylation. In one preferred embodiment, consensus substrate peptides are altered by introduction of alanine for phosphorylation targets serine or threonine.

[0025] In another preferred embodiment, the present invention relates to a method of inhibiting Raf kinase activity comprising replacing a codon within the Raf gene encoding a serine or threonine amino acid for a codon encoding an amino acid not suceptable to phosphorylation and transforming said gene into a cell such that said Raf activity is inhibited.

[0026] In yet another embodiment, the present invention relates to a method of inhibiting Raf kinase activity comprising modifying Raf by replacing a serine or threonine amino acid within Raf for an amino acid not suceptable to phosphorylation and delivering said modified Raf to a cell such that said Raf expression is inhibited.

[0027] The present invention is described in further detail in the following non-limiting examples (see Kolch W. et al (1991) Nature 349:426 and App et al. (1991) Molec. Cell. Biol. 11(2):913-919).

EXAMPLES

[0028] The following protocols and experimental details are referenced in the Examples that follow:

[0029] Expression plasmid construction. pMNC digested with XhoI and BamHl was blunt ended with T4 DNA polymerase. The mouse and human cDNAs were cut with StuI or HincII, respectively, and appropriate sized fragments were ligated with the pMNC vector. GMA contains residues 1254-1426 and GMS 1427-1697 of the mouse cDNA, HCR 1-903 of the human c-raf-1 cDNA (Bonner et al. Nucleic Acids Res. 14:1009-1015 (1986)). The translation termination codon for HCR sense is provided by vector sequences resulting in the addition of nine amino acids. To construct p301-1 (sense orientation), an EcoRI-Xbal fragment of p628 (Bonner et al. Nucleic Acids Res. 14:1009-1015 (1986)) encompassing the coding sequence of a human c-raf-1 cDNA was cloned into BluescriptKS (Stratagene). Lysine(375) was changed to tryptophan by site-directed mutagenesis resulting in the creation of a unique BamHI site. This cDNA was transferred into the Sacl-XhoI sites of pSVL (Pharmacia), then cloned into the Xhol-BamHI sites of pMNC as an XhoI-BamHI (partial digest) fragment. The corresponding antisense plasmid, p301-2, was generated by cloning the blunt-ended c-raf 301 EcoRi-Xbal fragment into blunt-ended pMNC.

[0030] Western analysis with PBB1. Cells were lysed in TBST (160 mM NaCl, 20 mM Tris HCl, pH7.5, 2 mM EDTA, 1% Triton X-100, 1 mM PMSF). Lysates were adjusted to equal protein concentrations (Biorad protein assay kit). Raf proteins were precipitated with the monoclonal antibody PBB1 and analyzed by western blotting with the polyclonal serum #137 as described previously (Kolch, W. et al. Oncogene 6:713-720 (1990)).

[0031] Mitogen responsiveness and proliferative capacity assay. 10⁴ cells were plated on cover slips and serum-starved for 24 h before incubation with 20% fetal calf serum (Gibco) or 100 ng ml⁻¹ TPA (Sigma). 14 h after addition of mitogens, cells were labelled with 1 μCiml⁻¹ ³H-thymidine for 5 h. Cells were counter-stained with Giemsa and labelled nuclei were counted. For long-term growth curves. 10⁵ cells were seeded in six-well plates in DMEM medium supplemented with 10% FCS. Each day, one well was trypsinized and counted with a Coulter cell counter. All determinations were performed in triplicate.

[0032] Cell maintenance. NIH 3T3 clone 2.2 cells devoid of endogenous EGF-R were transfected with wild-type (HER14) or kinase-negative (DK721A) receptors as described previously (Honegger, A. M., et al. (1987) Cell 51:199-209; Honegger, A. M. (1987) Mol. Cell. Biol. 7:4567-4571; Margolis, B., et al. (1989) Cell 57:1101-1107). In the case of the kinase-negative receptor mutant, the putative ATP binding lysine was substituted by an alanine (Honegger, A. M., et al. (1987) Cell 51:199-209; Honegger, A. M. (1987) Mol. Cell. Biol. 7:4567-4571). Cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 10% (vol/vol) calf serum.

[0033] Preparations of cytosolic cell extracts and immunoprecipitations. Cells were grown in 75 cm² flasks in DMEM containing 10% calf serum until confluency and starved overnight in 0.05% calf serum. Before lysis, cells were exposed to 40 nM EGF for 0 min at 37° C. and rinsed three times in phosphate-buffered saline. Control cells were not exposed to EGF. Cells were lysed in TBST buffer (50 mM Tris hydrochloride (pH 7.3), 150 mM NaCl, 0.5% Triton X-100) or in RIPA buffer (50 mM Tris hydrochloride (pH 7.3), 150 mM NaCl, 1% Triton X-100, 0.5% desoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 1 mM dithiothreitol, 0.2 mM sodium orthovanadate, 25 mM sodium fluoride, 10 mM sodium pyrophosphate, 25 mM glycerophosphate). Insoluble material was removed by centrifugation at 4° C.; for 30 min at 12,000×g. Protein concentrations were determined by the method of Bradford (Bradford, M. M. (1976) Anal. Bichem. 72:248-254). Immunoprecipitations were performed by incubating lysates with polyclonal rabbit antiserum against the v-Raf 30-kDa protein (Kolch, W., et al. (1988) Biochim. Biophys. Acta 949:233-239) or a polyclonal rabbit antiserum against a synthetic peptide (SP63) corresponding to the last 12 carboxy-terminal amino acids of the Raf-1 protein and protein A for 3 h at 4° C.

[0034] Western immunoblotting. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE). The gels were electroblotted on nitrocellulose, and the blots were blocked with 5% (wt/vol) gelatin in TBST buffer and incubated with polyclonal antiserum against Raf-1 or EGF-R. After extensive washing with TBST buffer, the blot was labeled with ¹²⁵J-staph protein A (Dupont NEN). Nonbound ¹²⁵J-staph was removed by washing the blots with TBST buffer, and the dried membrane was exposed to x-ray film.

[0035] Immunocomplex kinase assay. Immunoprecipitates were washed three times with cold RIPA buffer and twice with kinase buffer (50 mM Tris hydrochloride (pH 7.3), 150 mM NaCl, 12.5 mM MnCl₂, 1 mM dithiothreitol, and 0.2% Tween 20). Immunocomplex kinase assays were performed by incubating immunoprecipitates from 10⁶ cells in 80 μl of kinase buffer with 20 μCi of [γ-³²P]ATP (10 mCi/ml) and 20 μl of the Raf-1 substrate peptide (5 mg/ml) for 30 min at 25° C. The sequence of the Raf-1 substrate peptide is IVQQFGFQRRASDDGKLTD. A control peptide had tyrosine in position 5, as does wild-type Raf-1, and alanine in place of serine in position 12. The assay was linear for at least 40 min. The phosphorylation reaction was terminated by spotting 15-μl aliquots of the assay mixture on a 2- by 2-cm Whatman P81 phosphocellulose filter. The filters were washed four times for 30 min in 1% orthophosphoric acid and air dried, and the amount of ³²P incorporated was determined by the Cerenkov method. No differences were observed when counts were compared between filters on which the whole reaction mix or only the supernatant was spotted. Peptide phosphorylation in this assay was verified by running the reaction products on 20% SDS gels.

[0036] Phosphoamino acid analysis. One-dimensional phosphoamino acid analysis was carried out as described by Cooper et al. (Cooper, A. A., et al. (1983) Methods in Enzymol. 99:387-402). Phosphoamino acids were separated at pH 1.8 (6% formic acid and 15% acetic acid) for 4 h at 750 V.

EXAMPLE 1 Antisense Experiments

[0037] Portions of c-raf-1 cDNAs were expressed in sense and antisense orientation using the pMNC vector (FIG. 1). After transfection into NIH/3T3 cells the number of neomycin-resistant colonies was scored. Antisense constructs yielded roughly half the number of colonies as did the corresponding sense construct or the vector control, indicating that raf antisense RNA interferes with viability and/or proliferation. As NIH/3T3 cells express no B-raf and 10-fold less A-raf than Raf-1 (Storm, S. M. et al. Oncogene 5:345-351 (1990)), the effect can be ascribed to interference with Raf-1. Antisense colonies were generally smaller and grew slower than sense or vector control colonies. Ten out of ten antisense colonies showed barely detectable levels of Raf-1 protein, whereas levels in sense control clones were unchanged. An alternative approach to Raf-1 inhibition used inactive mutants (Rapp, U. R. et al. The Oncogene Handbook (eds. E. P. Reddy, A. M. Skalka and T. Curran) 213-253 (Elsevier Science. The Netherlands, 1988); Heidecker, G. et al. Molec. Cell. Biol. 10:2503-2512 (1990)). A truncated Raf-1 protein (HCR) corresponding to conserved region 1 reduced colony numbers fourfold. A kinase-defective Raf-1 mutant protein, craf301 (plasmid p301-1), was even more efficient, decreasing colony yield about sevenfold (Heidecker, G. et al. Molec. Cell. Biol. 10:2503-2512 (1990)). The surviving colonies from these experiments could not be maintained as stable cell lines. raf-transformed cell-lines were examined for morphological reversion and inhibition of proliferation. p301 constructs were transfected into 208-F12 fibroblasts which overexpress a transforming mouse Raf-1 protein (Schultz, A. M. et al. Oncogene 2:187-193 (1988)). p301-2 caused partial or complete reversion of the transformed phenotype in approximately half the transfectants. Reversion correlated with loss of anchorage-independent growth. Again, p301-1 was more efficient than p301-2 (FIG. 2a). These clones were unstable, but cell-lines sufficiently stable for biochemical analysis were obtained after pGMS transfection of v-raf transformed cells, F4 (Rapp, U. R., et al. Proc. Natl. Acad. Sci. U.S.A. 80:4218-4222 (1983)). Neither pMNC nor the control plasmid GMS-7 was effective, whereas the antisense construct, GMS-8, completely or partially reverted F4 (FIG. 2b). Reduction of raf mRNA and protein levels correlated with the extent of reversion (FIG. 2c). In one clone, GMS-8/3 (marked f* in FIG. 2c), raf protein expression was undetectable. These cells grew extremely slowly, arresting at 50-60% confluency, and eventually died.

[0038] To measure the effects of raf-protein depletion on the mitogen response, the ability of serum and TPA to induce DNA synthesis in serum-starved cells was determined (FIG. 3a). F4 and GMS-7 cells synthesize DNA independently of mitogens. Constitutive DNA synthesis was diminished in GMS-8/2, which retained an inducible response similar to NIH/3T3 cells. GMS-8/3 was completely blocked in constitutive and TPA-inducible DNA replication. Serum-stimulation of GMS-8/3 was reduced seven-fold, and long-term growth was also severely diminished (FIG. 3b).

[0039] v-Ki-ras-transformed NIH/3T3 cells were transfected with the p301 plasmids (Table 1a). p301-1 and p301-2 reduced neomycin (neo)-resistant colony yield to a similar degree as in NIH/3T3 cells (FIG. 1), suggesting that Raf-1 is required for proliferation of raf-transformed cells. Morphological reversion of established ras-transformed cells was less efficient than of raf-transformed cells (FIG. 2). To test the effect of raf-inhibition on the initiation of ras-transformation, a constant amount of v-Ha-ras (pSV2neo/ras, Clanton, D. J. et al. Molec. Cell. Biol. 7:3092-3097 (1987)) plasmid was co-transfected with an equal or four-molar amount of the p301 vectors (Table 1b). Although the neomycin resistance of pMNC-based plasmids accounted for a background of flat neo• colonies that presumably did not express pSV2neo/ras, transfection with p301 vectors markedly increased the number of morphological revertants at the expense of transformed colonies. The inhibition was dose-dependent and almost complete at four-molar excess of p301-1.

[0040] Thus, NIH/3T3 cells RAf-1 kinase functions downstream of membrane receptors and ras proteins and is essential for growth-induction by serum factors and protein kinase C. Membrane receptor systems can now be examined individually for Raf-1-dependence by inhibition with the blocking constructs described herein. Furthermore, the proposed position of raf in the communication pathway between cell membrane and nucleus makes raf an attractive target for the design of novel antiproliferative agents, especially as this data show that raf inhibition is dominant over transformation by ras and by implication by other non-nuclear oncogenes. TABLE I raf-inhibition blocks ras-mediated proliferation and transformation a) v-Ki-ras cell trasfections Yield of nco¹ Morphology of nco¹ colonies Plasmids colonies flat intermediate transformed pMNC¹ 100 ± 0%  0 ± 0%  0 ± 1% 100 ± 1% pMNC301-2²  61 ± 8%  2 ± 1% 15 ± 7%  83 ± 7% pMNC301-1³  30 ± 7% 15 ± 3% 15 ± 5%  70 ± 6% b) NIH/3T3 co-transfection with v-Ha-ras (pSV2nco/ras) and p301 Plasmids Morphology of nco¹ colonies (ratio 1:1) inhibition⁴ flat intermediate transformed ras + pMNC¹  0 ± 3% 27 ± 3% 17 ± 5% 56 ± 3% ras + p301-2² 53 ± 4% 28 ± 1% 46 ± 9% 26 ± 7% ras + p301-1³ 61 ± 3% 46 ± 4% 32 ± 7% 22 ± 4% Plasmids (ratio 1:4) ras + pMNC¹  0 ± 1% 33 ± 4% 23 ± 3% 44 ± 3% ras + p301-2² 61 ± 5% 48 ± 5% 35 '5 7% 17 ± 4$ ras + p301-1³ 84 ± 4% 67 ± 4% 25 ± 6%  7 ± 3%

EXAMPLE 2 Association and Kinase Activity of Raf-1 with the EGF Receptor

[0041] To determine whether EGF induces the shift in migration in SDS gels that is typical for phosphorylation activation of Raf-1 protein kinase, lysates of treated and control cells were subjected to immunoprecipitation and immunoblotting with Raf-1-specific antiserum. NIH 3T3 cells lacking endogenous EGF-R but expressing approximately 3×10⁵ human wild-type (HER14) EGF-R or kinase-negative mutant K721A EGF-R were transferred to starvation medium (0.05% calf serum) at early confluency and stimulated with EGF at 40 nM for 0 to 10 min. The effect of EGF on Raf-1 mobility is shown in FIG. 4. In the absence of EGF treatment, Raf-1 migrates as a single polypetide of 72 kDa, corresponding to the expected molecular mass of Raf-1 protein kinase (Bonner, T. I., et al. (1986) Nucleic Acids Res. 14:1009-1015). The addition of EFG to HER14 but not to K721A cells resulted in a small increase in apparent mass of Raf-1 to 74 kDa. This shift first became detectable by 5 min, when approximately 50% of Raf-1 protein was affected, and continued to spread so that by 10 min the entire pool of Raf-1 protein had been modified. The inability of EFG to induce the Raf mobility shift in NIH 3T3 cells expressing the kinase-negative mutant of EGF-R demonstrates that receptor dimerization is not sufficient for Raf-1 modification, since the point mutation in K721A does not affect this event (Ulrich, A., et al. (1990) Cell 61:203-212). It therefore seemed likely that the kinase activity of the EGF-R was important in mediating induction of the mobility shift in Raf-1.

[0042] The increase in apparent molecular mass of Raf-1 protein upon EGF treatment was due to phosphorylation, since incubation with potato acid phosphatase completely reversed the gel retardation. To evaluate the effect of EFG-stimulated raf-1 protein phosphorylation on its serine- and threonine-specific protein kinase activity, immune complex kinase assays were performed that utilized a synthetic peptide (IVQQFGFQRRASDDGKLTD) or histone H1 as a substrate. The peptide corresponds to a potential autophosphorylation site in the Raf-1 kinase, which has been altered by substitution of phenylalanine for tyrosine in position 7 so as to restrict it from tyrosine phosphorylation.

[0043] For kinase assays, lysates of HER14 and K721A cells were prepared before and after stimulation with 40 nM EFG for 10 min. Comparison of the levels of kinase activity in Raf-containing immunoprecipitates showed a sixfold stimulation in HER14 cells upon EFG treatment (FIG. 5). Similar data were obtained when histone H1 was used as a substrate. Consistent with the absence of the EGF-induced mobility shift of Raf-1 in NIH 3T3 cells expressing the kinase-negative mutant for of the EGF-R, no stimulation of Raf-1 protein kinase activity was observed in K721A cells (FIG. 5). When Raf-1 kinase activity was assayed with a modified version of the substrate peptide in which Ser-12 was replaced by alanine and Tyr-5 was retained, no counts were detected on the spotted filters. This indicates that the kinase activity measured by the assay did not include a contribution of a contaminating tyrosine kinase activity.

[0044] Activity EGF-R associates with the candidate signal transducing enzyme PLC_(γ) (Margolis, B., et al. (1990) Mol. Cell. Biol. 10:435-441; Margolis, B., et al. (1989) Cell 57:1101-1107; Meisenhelder, J., et al. (1989) Cell 57:1109-1112; Wahl, M., et al. (1989) Proc. Natl. Acad. Sci. USA 86:1568-1572). Similarly, Raf-1 was shown to coimmunoprecipitate with activated PDGF-β receptor in cell lines expressing high levels of receptors (Morrison, D. K., et al. (1989) Cell 58:648-657). To evaluate whether ligand-induced activation of Raf-1 protein kinase by the EGF-R correlated with receptor association, two cell systems were used: the NIH 3T3 cells expressing wild-type and mutant receptors (FIG. 6A) and human A431 cells (FIG. 6B) expressing approximately 2×10⁶ EGF-R per cell (15, 16). Serum-starved cells were stimulated with 40 nM EGF for 10 min. and lysates from cells were immunoprecipitated with Raf-1 or EGF-R specific antibodies. After separation by SDS-PAGE and transfer to nitrocellulose, immunoblotting was performed with either anti-EGF-R or anti-Raf-1 antibodies. EGF-R is present in anti-Raf-1 antibody immunoprecipitates from EGF-treated cells (FIG. 6). The coprecipitating EGF-R in HER14 cells has a decreased mobility on PAGE, compared with that of the EGF-R from untreated controls (FIG. 6A, lanes 4 and 6); this decreased mobility was previously demonstrated to be due to ligand-induced autophosphorylation (Margolis, B., et al. (1989) Cell 57:1101-1107). Cells expressing the kinase-negative mutant receptor KD712A did not show the mobility shift in the EGF-R upon EGF treatment and lacked EGF stimulation of EGF-R Raf-1 coimmunoprecipitation. A small amount of unshifted EGF-R was detected in Raf-1 immunoprocipitates from all cells; this EGF-R could be reduced by preclearing with preimmune serum. EGF-R can be coprecipitated in lysates from EGF-treated A431 cells, whereas there is not EGF-R present in immunoprecipitates from untreated cells (FIG. 6). Sequential reprobing of the Western blot with polyclonal Raf-1 rabbit antiserum (FIG. 6B) indicates that a small fraction (˜1%) of the EGF-R associates with shifted Raf-1. Furthermore, the blot demonstrates that the EGF-R-Raf-1 association was not due to unequal loading of the gel with Raf-1 immunoprecipitates. Estimates from three independent experiments indicate that the fraction of immunoprecipitable EGF-R protein that is present in Raf-1 antibody precipitates from EGF-treated HER14 or A431 cells is on the order of 1.0%.

[0045] Considering the observed association of Raf-1 protein with activated EGF-R as well as the EGF-induced mobility shift of Raf-1, it might be expected that the receptor-associated fraction of Raf-1 was phosphorylated on tyrosine. The immunoblots from experiments in FIG. 6 were therefore reprobed with antiphosphotyrosine antibodies. The antibodies readily detected EFG-induced tyrosine phosphorylation of the EGF-R, PLCτ, GAP, and other unknown substrates (Ulrich, A., et al. (1990) Cell 61:203-212), but no tyrosine phosphorylated bands in the size range of Raf-1 protein were detected. The experiment was scaled up to examine the presence of tyrosine-phosphorylated Raf-1 protein in anti-Raf or anti-EGF-R antibody immunoprecipiates from 10⁶ HER14 cells per lane; again, tyrosine phosphorylation of Raf-1 could not be detected. Consistent with the absence of anti-phosphotyrosine antibody-reactive Raf-1 protein, phosphoamino acid analysis of Raf-1 from EGF-treated cells did not reveal any phosphotyrosine (FIG. 7). For this experiment, 10⁷ HER14 cells were labeled with ³²Pi, and the Raf-1 proteins were immunoprecipitated with anti-v-Raf 30-kDa polyclonal antiserum and subjected to SDS-PAGE. Phospholabeled Raf-1 protein was excised from the gel, electroeluted, and hydrolyzed in 6 N HCl. The only labeled phosphoamino acid detectable was phosphoserine; thus it can be concluded that EGF induced an increase in serine phosphorylation of c-Raf (FIG. 7). When the same experiment was done with A431 cells, trace amounts of phosphotyrosine were detected that were independent of EGF treatment. The lower limit for detection of phosphotyrosine in Raf-1 in these experiments was on the order of 1% of phosphoserine.

[0046] The absence of tyrosine phosphorylation of Raf-1 protein in response to EGF in HER14 cells raises the possibility that serine protein kinase(s) acts as an intermediate in a kinase cascade connecting the stimulated EGF-R to activation of Raf-1 kinase. One candidate for this role is PKC, since this enzyme has previously been shown, upon treatment of cells with tetradecanoylphorbol-13-acetate (TPA), to trigger Raf-1 phosphorylation and kinase activation (Morrison, D. K., et al. (1988) Proc. Natl. Acad. Sci. USA 85:8855-8859; Siegel, J. N., et al. (1990) J. Biol. Chem. 265:18472-18480). It was therefore examined whether EGF induction of the Raf-1 mobility shift was dependent on the presence of PKC (FIG. 8). HER14 cells were pretreated with 200 ng of TPA for 72 h for complete downregulation of PKC and then tested for their ability to respond to EGF with Raf-1 retardation. The PKC down-regulation by pretreatment with TPA was effective in eliminating the TPA-induced Raf-1 retardation. In contrast, EGF-induced Raf-1 mobility shift was not blocked by down-regulation of PKC.

[0047] All publications mentioned hereinabove are hereby incorporated in their entirety by reference. In particular, Kolch W et al (1991) Nature 349:426-428 and App H et al (1991) Molecular and Cellular Biology 11(2):913-919 are hereby incorporated in their entirety by reference.

[0048] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.

1 8 19 amino acids amino acid single linear peptide 1 Ile Val Gln Gln Phe Gly Phe Gln Arg Arg Ala Ser Asp Asp Gly Leu 1 5 10 15 Lys Thr Asp 2975 base pairs nucleic acid single linear DNA (genomic) 2 CCGAATGTGA CCGCCTCCGC TCCCTACCCG CCGCGGGGAG GAGGAGCGGG CGAGAACGTG 60 CCGCCGAACG ACAGGACGTT GGGGCGGCCT GGCTCCCTCA GGTTTAAGAA TTGTTTAAGC 120 TGCATCAATG GAGCACATAC AGGGAGCTTG GAAGACGATC AGCAATGGTT TTGGATTCAA 180 AGATGCCGTG TTTGATGGCT CCAGCTGCAT CTCTCCTACA ATAGTTCAGC AGTTTGGCTA 240 TCAGCGCCGG GCATCAGATG ATGGCAAACT CACAGATCCT TCTAAGACAA GCAACACTAT 300 CCGTGTTTTC TTGCCGAACA AGCAAAGAAC AGTGGTCAAT GTGCGAAATG GAATGAGCTT 360 GCATGACTGC CTTATGAAAG CACTCAAGGT GAGGGGCCTG CAACCAGAGT GCTGTGCAGT 420 GTTCAGACTT CTCCACGAAC ACAAAGGTAA AAAAGCACGC TTAGATTGGA ATACTGATGC 480 TGCGTCTTTG ATTGGAGAAG AACTTCAAGT AGATTTCCTG GATCATGTTC CCCTCACAAC 540 ACACAACTTT GCTCGGAAGA CGTTCCTGAA GCTTGCCTTC TGTGACATCT GTCAGAAATT 600 CCTGCTCAAT GGATTTCGAT GTCAGACTTG TGGCTACAAA TTTCATGAGC ACTGTAGCAC 660 CAAAGTACCT ACTATGTGTG TGGACTGGAG TAACATCAGA CAACTCTTAT TGTTTCCAAA 720 TTCCACTATT GGTGATAGTG GAGTCCCAGC ACTACCTTCT TTGACTATGC GTCGTATGCG 780 AGAGTCTGTT TCCAGGATGC CTGTTAGTTC TCAGCACAGA TATTCTACAC CTCACGCCTT 840 CACCTTTAAC ACCTCCAGTC CCTCATCTGA AGGTTCCCTC TCCCAGAGGC AGAGGTCGAC 900 ATCCACACCT AATGTCCACA TGGTCAGCAC CACGCTGCCT GTGGACAGCA GGATGATTGA 960 GGATGCAATT CGAAGTCACA GCGAATCAGC CTCACCTTCA GCCCTGTCCA GTAGCCCCAA 1020 CAATCTGAGC CCAACAGGCT GGTCACAGCC GAAAACCCCC GTGCCAGCAC AAAGAGAGCG 1080 GGCACCAGTA TCTGGGACCC AGGAGAAAAA CAAAATTAGG CCTCGTGGAC AGAGAGATTC 1140 AAGCTATTAT TGGGAAATAG AAGCCAGTGA AGTGATGCTG TCCACTCGGA TTGGGTCAGG 1200 CTCTTTTGGA ACTGTTTATA AGGGTAAATG GCACGGAGAT GTTGCAGTAA AGATCCTAAA 1260 GGTTGTCGAC CCAACCCCAG AGCAATTCCA GGCCTTCAGG AATGAGGTGG CTGTTCTGCG 1320 CAAAACACGG CATGTGAACA TTCTGCTTTT CATGGGGTAC ATGACAAAGG ACAACCTGGC 1380 AATTGTGACC CAGTGGTGCG AGGGCAGCAG CCTCTACAAA CACCTGCATG TCCAGGAGAC 1440 CAAGTTTCAG ATGTTCCAGC TAATTGACAT TGCCCGGCAG ACGGCTCAGG GAATGGACTA 1500 TTTGCATGCA AAGAACATCA TCCATAGAGA CATGAAATCC AACAATATAT TTCTCCATGA 1560 AGGCTTAACA GTGAAAATTG GAGATTTTGG TTTGGCAACA GTAAAGTCAC GCTGGAGTGG 1620 TTCTCAGCAG GTTGAACAAC CTACTGGCTC TGTCCTCTGG ATGGCCCCAG AGGTGATCCG 1680 AATGCAGGAT AACAACCCAT TCAGTTTCCA GTCGGATGTC TACTCCTATG GCATCGTATT 1740 GTATGAACTG ATGACGGGGG AGCTTCCTTA TTCTCACATC AACAACCGAG ATCAGATCAT 1800 CTTCATGGTG GGCCGAGGAT ATGCCTCCCC AGATCTTAGT AAGCTATATA AGAACTGCCC 1860 CAAAGCAATG AAGAGGCTGG TAGCTGACTG TGTGAAGAAA GTAAAGGAAG AGAGGCCTCT 1920 TTTTCCCCAG ATCCTGTCTT CCATTGAGCT GCTCCAACAC TCTCTACCGA AGATCAACCG 1980 GAGCGCTTCC GAGCCATCCT TGCATCGGGC AGCCCACACT GAGGATATCA ATGCTTGCAC 2040 GCTGACCACG TCCCCGAGGC TGCCTGTCTT CTAGTTGACT TTGCACCTGT CTTCAGGCTG 2100 CCAGGGGAGG AGGAGAAGCC AGCAGGCACC ACTTTTCTGC TCCCTTTCTC CAGAGGCAGA 2160 ACACATGTTT TCAGAGAAGC TCTGCTAAGG ACCTTCTAGA CTGCTCACAG GGCCTTAACT 2220 TCATGTTGCC TTCTTTTCTA TCCCTTTGGG CCCTGGGAGA AGGAAGCCAT TTGCAGTGCT 2280 GGTGTGTCCT GCTCCCTCCC CACATTCCCC ATGCTCAAGG CCCAGCCTTC TGTAGATGCG 2340 CAAGTGGATG TTGATGGTAG TACAAAAAGC AGGGGCCCAG CCCCAGCTGT TGGCTACATG 2400 AGTATTTAGA GGAAGTAAGG TAGCAGGCAG TCCAGCCCTG ATGTGGAGAC ACATGGGATT 2460 TTGGAAATCA GCTTCTGGAG GAATGCATGT CACAGGCGGG ACTTTCTTCA GAGAGTGGTG 2520 CAGCGCCAGA CATTTTGCAC ATAAGGCACC AAACAGCCCA GGACTGCCGA GACTCTGGCC 2580 GCCCGAAGGA GCCTGCTTTG GTACTATGGA ACTTTTCTTA GGGGACACGT CCTCCTTTCA 2640 CAGCTTCTAA GGTGTCCAGT GCATTGGGAT GGTTTTCCAG GCAAGGCACT CGGCCAATCC 2700 GCATCTCAGC CCTCTCAGGA GCAGTCTTCC ATCATGCTGA ATTTTGTCTT CCAGGAGCTG 2760 CCCCTATGGG GCGGGCCGCA GGGCCAGCCT GTTTCTCTAA CAAACAAACA AACAAACAGC 2820 CTTGTTTCTC TAGTCACATC ATGTGTATAC AAGGAAGCCA GGAATACAGG TTTTCTTGAT 2880 GATTTGGGTT TTAATTTTGT TTTTATTGCA CCTGACAAAA TACAGTTATC TGATGGTCCC 2940 TCAATTATGT TATTTTAATA AAATAAATTA AATTT 2975 648 amino acids amino acid single linear peptide 3 Met Glu His Ile Gln Gly Ala Trp Lys Thr Ile Ser Asn Gly Phe Gly 1 5 10 15 Phe Lys Asp Ala Val Phe Asp Gly Ser Ser Cys Ile Ser Pro Thr Ile 20 25 30 Val Gln Gln Phe Gly Tyr Gln Arg Arg Ala Ser Asp Asp Gly Lys Leu 35 40 45 Thr Asp Pro Ser Lys Thr Ser Asn Thr Ile Arg Val Phe Leu Pro Asn 50 55 60 Lys Gln Arg Thr Val Val Asn Val Arg Asn Gly Met Ser Leu His Asp 65 70 75 80 Cys Leu Met Lys Ala Leu Lys Val Arg Gly Leu Gln Pro Glu Cys Cys 85 90 95 Ala Val Phe Arg Leu Leu His Glu His Lys Gly Lys Lys Ala Arg Leu 100 105 110 Asp Trp Asn Thr Asp Ala Ala Ser Leu Ile Gly Glu Glu Leu Gln Val 115 120 125 Asp Phe Leu Asp His Val Pro Leu Thr Thr His Asn Phe Ala Arg Lys 130 135 140 Thr Phe Leu Lys Leu Ala Phe Cys Asp Ile Cys Gln Lys Phe Leu Leu 145 150 155 160 Asn Gly Phe Arg Cys Gln Thr Cys Gly Tyr Lys Phe His Glu His Cys 165 170 175 Ser Thr Lys Val Pro Thr Met Cys Val Asp Trp Ser Asn Ile Arg Gln 180 185 190 Leu Leu Leu Phe Pro Asn Ser Thr Ile Gly Asp Ser Gly Val Pro Ala 195 200 205 Leu Pro Ser Leu Thr Met Arg Arg Met Arg Glu Ser Val Ser Arg Met 210 215 220 Pro Val Ser Ser Gln His Arg Tyr Ser Thr Pro His Ala Phe Thr Phe 225 230 235 240 Asn Thr Ser Ser Pro Ser Ser Glu Gly Ser Leu Ser Gln Arg Gln Arg 245 250 255 Ser Thr Ser Thr Pro Asn Val His Met Val Ser Thr Thr Leu Pro Val 260 265 270 Asp Ser Arg Met Ile Glu Asp Ala Ile Arg Ser His Ser Glu Ser Ala 275 280 285 Ser Pro Ser Ala Leu Ser Ser Ser Pro Asn Asn Leu Ser Pro Thr Gly 290 295 300 Trp Ser Gln Pro Lys Thr Pro Val Pro Ala Gln Arg Glu Arg Ala Pro 305 310 315 320 Val Ser Gly Thr Gln Glu Lys Asn Lys Ile Arg Pro Arg Gly Gln Arg 325 330 335 Asp Ser Ser Tyr Tyr Trp Glu Ile Glu Ala Ser Glu Val Met Leu Ser 340 345 350 Thr Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp 355 360 365 His Gly Asp Val Ala Val Lys Ile Leu Lys Val Val Asp Pro Thr Pro 370 375 380 Glu Gln Phe Gln Ala Phe Arg Asn Glu Val Ala Val Leu Arg Lys Thr 385 390 395 400 Arg His Val Asn Ile Leu Leu Phe Met Gly Tyr Met Thr Lys Asp Asn 405 410 415 Leu Ala Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr Lys His 420 425 430 Leu His Val Gln Glu Thr Lys Phe Gln Met Phe Gln Leu Ile Asp Ile 435 440 445 Ala Arg Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys Asn Ile 450 455 460 Ile His Arg Asp Met Lys Ser Asn Asn Ile Phe Leu His Glu Gly Leu 465 470 475 480 Thr Val Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg Trp 485 490 495 Ser Gly Ser Gln Gln Val Glu Gln Pro Thr Gly Ser Val Leu Trp Met 500 505 510 Ala Pro Glu Val Ile Arg Met Gln Asp Asn Asn Pro Phe Ser Phe Gln 515 520 525 Ser Asp Val Tyr Ser Tyr Gly Ile Val Leu Tyr Glu Leu Met Thr Gly 530 535 540 Glu Leu Pro Tyr Ser His Ile Asn Asn Arg Asp Gln Ile Ile Phe Met 545 550 555 560 Val Gly Arg Gly Tyr Ala Ser Pro Asp Leu Ser Lys Leu Tyr Lys Asn 565 570 575 Cys Pro Lys Ala Met Lys Arg Leu Val Ala Asp Cys Val Lys Lys Val 580 585 590 Lys Glu Glu Arg Pro Leu Phe Pro Gln Ile Leu Ser Ser Ile Glu Leu 595 600 605 Leu Gln His Ser Leu Pro Lys Ile Asn Arg Ser Ala Ser Glu Pro Ser 610 615 620 Leu His Arg Ala Ala His Thr Glu Asp Ile Asn Ala Cys Thr Leu Thr 625 630 635 640 Thr Ser Pro Arg Leu Pro Val Phe 645 2458 base pairs nucleic acid single linear DNA (genomic) 4 TGACCCAATA AGGGTGGAAG GCTGAGTCCC GCAGAGCCAA TAACGAGAGT CCGAGAGGCG 60 ACGGAGGCGG ACTCTGTGAG GAAACAAGAA GAGAGGCCCA AGATGGAGAC GGCGGCGGCT 120 GTAGCGGCGT GACAGGAGCC CCATGGCACC TGCCCAGCCC CACCTCAGCC CATCTTGACA 180 AAATCTAAGG CTCCATGGAG CCACCACGGG GCCCCCCTGC CAATGGGGCC GAGCCATCCC 240 GGGCAGTGGG CACCGTCAAA GTATACCTGC CCAACAAGCA ACGCACGGTG GTGACTGTCC 300 GGGATGGCAT GAGTGTCTAC GACTCTCTAG ACAAGGCCCT GAAGGTGCGG GGTCTAAATC 360 AGGACTGCTG TGTGGTCTAC CGACTCATCA AGGGACGAAA GACGGTCACT GCCTGGGACA 420 CAGCCATTGC TCCCCTGGAT GGCGAGGAGC TCATTGTCGA GGTCCTTGAA GATGTCCCGC 480 TGACCATGCA CAATTTTGTA CGGAAGACCT TCTTCAGCCT GGCGTTCTGT GACTTCTGCC 540 TTAAGTTTCT GTTCCATGGC TTCCGTTGCC AAACCTGTGG CTACAAGTTC CACCAGCATT 600 GTTCCTCCAA GGTCCCCACA GTCTGTGTTG ACATGAGTAC CAACCGCCAA CAGTTCTACC 660 ACAGTGTCCA GGATTTGTCC GGAGGCTCCA GACAGCATGA GGCTCCCTCG AACCGCCCCC 720 TGAATGAGTT GCTAACCCCC CAGGGTCCCA GCCCCCGCAC CCAGCACTGT GACCCGGAGC 780 ACTTCCCCTT CCCTGCCCCA GCCAATGCCC CCCTACAGCG CATCCGCTCC ACGTCCACTC 840 CCAACGTCCA TATGGTCAGC ACCACGGCCC CCATGGACTC CAACCTCATC CAGCTCACTG 900 GCCAGAGTTT CAGCACTGAT GCTGCCGGTA GTAGAGGAGG TAGTGATGGA ACCCCCCGGG 960 GGAGCCCCAG CCCAGCCAGC GTGTCCTCGG GGAGGAAGTC CCCACATTCC AAGTCACCAG 1020 CAGAGCAGCG CGAGCGGAAG TCCTTGGCCG ATGACAAGAA GAAAGTGAAG AACCTGGGGT 1080 ACCGGGATTC AGGCTATTAC TGGGAGGTAC CACCCAGTGA GGTGCAGCTG CTGAAGAGGA 1140 TCGGGACGGG CTCGTTTGGC ACCGTGTTTC GAGGGCGGTG GCATGGCGAT GTGGCCGTGA 1200 AGGTGCTCAA GGTGTCCCAG CCCACAGCTG AGCAGGCCCA GGCTTTCAAG AATGAGATGC 1260 AGGTGCTCAG GAAGACGCGA CATGTCAACA TCTTGCTGTT TATGGGCTTC ATGACCCGGC 1320 CGGGATTTGC CATCATCACA CAGTGGTGTG AGGGCTCCAG CCTCTACCAT CACCTGCATG 1380 TGGCCGACAC ACGCTTCGAC ATGGTCCAGC TCATCGACGT GGCCCGGCAG ACTGCCCAGG 1440 GCATGGACTA CCTCCATGCC AAGAACATCA TCCACCGAGA TCTCAAGTCT AACAACATCT 1500 TCCTACATGA GGGGCTCACG GTGAAGATCG GTGACTTTGG CTTGGCCACA GTGAAGACTC 1560 GATGGAGCGG GGCCCAGCCC TTGGAGCAGC CCTCAGGATC TGTGCTGTGG ATGGCAGCTG 1620 AGGTGATCCG TATGCAGGAC CCGAACCCCT ACAGCTTCCA GTCAGACGTC TATGCCTACG 1680 GGGTTGTGCT CTACGAGCTT ATGACTGGCT CACTGCCTTA CAGCCACATT GGCTGCCGTG 1740 ACCAGATTAT CTTTATGGTG GGCCGTGGCT ATCTGTCCCC GGACCTCAGC AAAATCTCCA 1800 GCAACTGCCC CAAGGCCATG CGGCGCCTGC TGTCTGACTG CCTCAAGTTC CAGCGGGAGG 1860 AGCGGCCCCT CTTCCCCCAG ATCCTGGCCA CAATTGAGCT GCTGCAACGG TCACTCCCCA 1920 AGATTGAGCG GAGTGCCTCG GAACCCTCCT TGCACCGCAC CCAGGCCGAT GAGTTGCCTG 1980 CCTGCCTACT CAGCGCAGCC CGCCTTGTGC CTTAGGCCCC GCCCAAGCCA CCAGGGAGCC 2040 AATCTCAGCC CTCCACGCCA AGGAGCCTTG CCCACCAGCC AATCAATGTT CGTCTCTGCC 2100 CTGATGCTGC CTCAGGATCC CCCATTCCCC ACCCTGGGAG ATGAGGGGGT CCCCATGTGC 2160 TTTTCCAGTT CTTCTGGAAT TGGGGGACCC CCGCCAAAGA CTGAGCCCCC TGTCTCCTCC 2220 ATCATTTGGT TTCCTCTTGG CTTTGGGGAT ACTTCTAAAT TTTGGGAGCT CCTCCATCTC 2280 CAATGGCTGG GATTTGTGGC AGGGATTCCA CTCAGAACCT CTCTGGAATT TGTGCCTGAT 2340 GTGCCTTCCA CTGGATTTTG GGGTTCCCAG CACCCCATGT GGATTTTGGG GGGTCCCTTT 2400 TGTGTCTCCC CCGCCATTCA AGGACTCCTC TCTTTCTTCA CCAAGAAGCA CAGAATTC 2458 606 amino acids amino acid single linear peptide 5 Met Glu Pro Pro Arg Gly Pro Pro Ala Asn Gly Ala Glu Pro Ser Arg 1 5 10 15 Ala Val Gly Thr Val Lys Val Tyr Leu Pro Asn Lys Gln Arg Thr Val 20 25 30 Val Thr Val Arg Asp Gly Met Ser Val Tyr Asp Ser Leu Asp Lys Ala 35 40 45 Leu Lys Val Arg Gly Leu Asn Gln Asp Cys Cys Val Val Tyr Arg Leu 50 55 60 Ile Lys Gly Arg Lys Thr Val Thr Ala Trp Asp Thr Ala Ile Ala Pro 65 70 75 80 Leu Asp Gly Glu Glu Leu Ile Val Glu Val Leu Glu Asp Val Pro Leu 85 90 95 Thr Met His Asn Phe Val Arg Lys Thr Phe Phe Ser Leu Ala Phe Cys 100 105 110 Asp Phe Cys Leu Lys Phe Leu Phe His Gly Phe Arg Cys Gln Thr Cys 115 120 125 Gly Tyr Lys Phe His Gln His Cys Ser Ser Lys Val Pro Thr Val Cys 130 135 140 Val Asp Met Ser Thr Asn Arg Gln Gln Phe Tyr His Ser Val Gln Asp 145 150 155 160 Leu Ser Gly Gly Ser Arg Gln His Glu Ala Pro Ser Asn Arg Pro Leu 165 170 175 Asn Glu Leu Leu Thr Pro Gln Gly Pro Ser Pro Arg Thr Gln His Cys 180 185 190 Asp Pro Glu His Phe Pro Phe Pro Ala Pro Ala Asn Ala Pro Leu Gln 195 200 205 Arg Ile Arg Ser Thr Ser Thr Pro Asn Val His Met Val Ser Thr Thr 210 215 220 Ala Pro Met Asp Ser Asn Leu Ile Gln Leu Thr Gly Gln Ser Phe Ser 225 230 235 240 Thr Asp Ala Ala Gly Ser Arg Gly Gly Ser Asp Gly Thr Pro Arg Gly 245 250 255 Ser Pro Ser Pro Ala Ser Val Ser Ser Gly Arg Lys Ser Pro His Ser 260 265 270 Lys Ser Pro Ala Glu Gln Arg Glu Arg Lys Ser Leu Ala Asp Asp Lys 275 280 285 Lys Lys Val Lys Asn Leu Gly Tyr Arg Asp Ser Gly Tyr Tyr Trp Glu 290 295 300 Val Pro Pro Ser Glu Val Gln Leu Leu Lys Arg Ile Gly Thr Gly Ser 305 310 315 320 Phe Gly Thr Val Phe Arg Gly Arg Trp His Gly Asp Val Ala Val Lys 325 330 335 Val Leu Lys Val Ser Gln Pro Thr Ala Glu Gln Ala Gln Ala Phe Lys 340 345 350 Asn Glu Met Gln Val Leu Arg Lys Thr Arg His Val Asn Ile Leu Leu 355 360 365 Phe Met Gly Phe Met Thr Arg Pro Gly Phe Ala Ile Ile Thr Gln Trp 370 375 380 Cys Glu Gly Ser Ser Leu Tyr His His Leu His Val Ala Asp Thr Arg 385 390 395 400 Phe Asp Met Val Gln Leu Ile Asp Val Ala Arg Gln Thr Ala Gln Gly 405 410 415 Met Asp Tyr Leu His Ala Lys Asn Ile Ile His Arg Asp Leu Lys Ser 420 425 430 Asn Asn Ile Phe Leu His Glu Gly Leu Thr Val Lys Ile Gly Asp Phe 435 440 445 Gly Leu Ala Thr Val Lys Thr Arg Trp Ser Gly Ala Gln Pro Leu Glu 450 455 460 Gln Pro Ser Gly Ser Val Leu Trp Met Ala Ala Glu Val Ile Arg Met 465 470 475 480 Gln Asp Pro Asn Pro Tyr Ser Phe Gln Ser Asp Val Tyr Ala Tyr Gly 485 490 495 Val Val Leu Tyr Glu Leu Met Thr Gly Ser Leu Pro Tyr Ser His Ile 500 505 510 Gly Cys Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly Tyr Leu Ser 515 520 525 Pro Asp Leu Ser Lys Ile Ser Ser Asn Cys Pro Lys Ala Met Arg Arg 530 535 540 Leu Leu Ser Asp Cys Leu Lys Phe Gln Arg Glu Glu Arg Pro Leu Phe 545 550 555 560 Pro Gln Ile Leu Ala Thr Ile Glu Leu Leu Gln Arg Ser Leu Pro Lys 565 570 575 Ile Glu Arg Ser Ala Ser Glu Pro Ser Leu His Arg Thr Gln Ala Asp 580 585 590 Glu Leu Pro Ala Cys Leu Leu Ser Ala Ala Arg Leu Val Pro 595 600 605 2229 base pairs nucleic acid single linear DNA (genomic) 6 GGGCAATATA TCTGGAGGCC TATGAAGAAT ACACCAGCAA GCTAGATGCA CTCCAACAAA 60 GAGAACAACA GTTATTGGAA TCTCTGGGGA ACGGAACTGA TTTTTCTGTT TCTAGCTCTG 120 CATCAATGGA TACCGTTACA TCTTCTTCCT CTTCTAGCCT TTCAGTGCTA CCTTCATCTC 180 TTTCAGTTTT TCAAAATCCC ACAGATGTGG CACGGAGCAA CCCCAAGTCA CCACAAAAAC 240 CTATCGTTAG AGTCTTCCTG CCCAACAAAC AGAGGACAGT GGTACCTGCA AGGTGTGGAG 300 TTACAGTCCG AGACAGTCTA AAGAAAGCAC TGATGATGAG AGGTCTAATC CCAGAGTGCT 360 GTGCTGTTTA CAGAATTCAG GATGGAGAGA AGAAACCAAT TGGTTGGGAC ACTGATATTT 420 CCTGGCTTAC TGGAGAAGAA TTGCATGTGG AAGTGTTGGA GAATGTTCCA CTTACAACAC 480 ACAACTTTGT ACGAAAAACG TTTTTCACCT TAGCATTTTG TGACTTTTGT CGAAAGCTGC 540 TTTTCCAGGG TTTCCGCTGT CAAACATGTG GTTATAAATT TCACCAGCGT TGTAGTACAG 600 AAGTTCCACT GATGTGTGTT AATTATGACC AACTTGATTT GCTGTTTGTC TCCAAGTTCT 660 TTGAACACCA CCCAATACCA CAGGAAGAGG CGTCCTTAGC AGAGACTGCC CTAACATCTG 720 GATCATCCCC TTCCGCACCC GCCTCGGACT CTATTGGGCC CCAAATTCTC ACCAGTCCGT 780 CTCCTTCAAA ATCCATTCCA ATTCCACAGC CCTTCCGACC AGCAGATGAA GATCATCGAA 840 ATCAATTTGG GCAACGAGAC CGATCCTCAT CAGCTCCCAA TGTGCATATA AACACAATAG 900 AACCTGTCAA TATTGATGAC TTGATTAGAG ACCAAGGATT TCGTGGTGAT GGAGGATCAA 960 CCACAGGTTT GTCTGCTACC CCCCCTGCCT CATTACCTGG CTCACTAACT AACGTGAAAG 1020 CCTTACAGAA ATCTCCAGGA CCTCAGCGAG AAAGGAAGTC ATCTTCATCC TCAGAAGACA 1080 GGAATCGAAT GAAAACACTT GGTAGACGGG ACTCGAGTGA TGATTGGGAG ATTCCTGATG 1140 GGCAGATTAC AGTGGGACAA AGAATTGGAT CTGGATCATT TGGAACAGTC TACAAGGGAA 1200 AGTGGCATGG TGATGTGGCA GTGAAAATGT TGAATGTGAC AGCACCTACA CCTCAGCAGT 1260 TACAAGCCTT CAAAAATGAA GTAGGAGTAC TCAGGAAAAC ACGACATGTG AATATCCTAC 1320 TCTTCATGGG CTATTCCACA AAGCCACAAC TGGCTATTGT TACCCAGTGG TGTGAGGGCT 1380 CCAGCTTGTA TCACCATCTC CATATCATTG AGACCAAATT TGAGATGATC AAACTTATAG 1440 ATATTGCACG ACAGACTGCA CAGGGCATGG ATTACTTACA CGCCAAGTCA ATCATCCACA 1500 GAGACCTCAA GAGTAATAAT ATATTTCTTC ATGAAGACCT CACAGTAAAA ATAGGTGATT 1560 TTGGTCTAGC TACAGTGAAA TCTCGATGGA GTGGGTCCCA TCAGTTTGAA CAGTTGTCTG 1620 GATCCATTTT GTGGATGGCA CCAGAAGTCA TCAGAATGCA AGATAAAAAT CCATACAGCT 1680 TTCAGTCAGA TGTATATGCA TTTGGAATTG TTCTGTATGA ATTGATGACT GGACAGTTAC 1740 CTTATTCAAA CATCAACAAC AGGGACCAGA TAATTTTTAT GGTGGGACGA GGATACCTGT 1800 CTCCAGATCT CAGTAAGGTA CGGAGTAACT GTCCAAAAGC CATGAAGAGA TTAATGGCAG 1860 AGTGCCTCAA AAAGAAAAGA GATGAGAGAC CACTCTTTCC CCAAATTCTC GCCTCTATTG 1920 AGCTGCTGGC CCGCTCATTG CCAAAAATTC ACCGCAGTGC ATCAGAACCC TCCTTGAATC 1980 GGGCTGGTTT CCAAACAGAG GATTTTAGTC TATATGCTTG TGCTTCTCCA AAAACACCCA 2040 TCCAGGCAGG GGGATATGGT GCGTTTCCTG TCCACTGAAA CAAATGAGTG AGAGAGTTCA 2100 GGAGAGTAGC AACAAAAGGA AAATAAATGA ACATATGTTT GCTTATATGT TAAATTGAAT 2160 AAAATACTCT CTTTTTTTTT AAGGTGGAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2220 AAAAAACCC 2229 650 amino acids amino acid single linear peptide 7 Met Asp Thr Val Thr Ser Ser Ser Ser Ser Ser Leu Ser Val Leu Pro 1 5 10 15 Ser Ser Leu Ser Val Phe Gln Asn Pro Thr Asp Val Ala Arg Ser Asn 20 25 30 Pro Lys Ser Pro Gln Lys Pro Ile Val Arg Val Phe Leu Pro Asn Lys 35 40 45 Gln Arg Thr Val Val Pro Ala Arg Cys Gly Val Thr Val Arg Asp Ser 50 55 60 Leu Lys Lys Ala Leu Met Met Arg Gly Leu Ile Pro Glu Cys Cys Ala 65 70 75 80 Val Tyr Arg Ile Gln Asp Gly Glu Lys Lys Pro Ile Gly Trp Asp Thr 85 90 95 Asp Ile Ser Trp Leu Thr Gly Glu Glu Leu His Val Glu Val Leu Glu 100 105 110 Asn Val Pro Leu Thr Thr His Asn Phe Val Arg Lys Thr Phe Phe Thr 115 120 125 Leu Ala Phe Cys Asp Phe Cys Arg Lys Leu Leu Phe Gln Gly Phe Arg 130 135 140 Cys Gln Thr Cys Gly Tyr Lys Phe His Gln Arg Cys Ser Thr Glu Val 145 150 155 160 Pro Leu Met Cys Val Asn Tyr Asp Gln Leu Asp Leu Leu Phe Val Ser 165 170 175 Lys Phe Phe Glu His His Pro Ile Pro Gln Glu Glu Ala Ser Leu Ala 180 185 190 Glu Thr Ala Leu Thr Ser Gly Ser Ser Pro Ser Ala Pro Ala Ser Asp 195 200 205 Ser Ile Gly Pro Gln Ile Leu Thr Ser Pro Ser Pro Ser Lys Ser Ile 210 215 220 Pro Ile Pro Gln Pro Phe Arg Pro Ala Asp Glu Asp His Arg Asn Gln 225 230 235 240 Phe Gly Gln Arg Asp Arg Ser Ser Ser Ala Pro Asn Val His Ile Asn 245 250 255 Thr Ile Glu Pro Val Asn Ile Asp Asp Leu Ile Arg Asp Gln Gly Phe 260 265 270 Arg Gly Asp Gly Gly Ser Thr Thr Gly Leu Ser Ala Thr Pro Pro Ala 275 280 285 Ser Leu Pro Gly Ser Leu Thr Asn Val Lys Ala Leu Gln Lys Ser Pro 290 295 300 Gly Pro Gln Arg Glu Arg Lys Ser Ser Ser Ser Ser Glu Asp Arg Asn 305 310 315 320 Arg Met Lys Thr Leu Gly Arg Arg Asp Ser Ser Asp Asp Trp Glu Ile 325 330 335 Pro Asp Gly Gln Ile Thr Val Gly Gln Arg Ile Gly Ser Gly Ser Phe 340 345 350 Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val Ala Val Lys Met 355 360 365 Leu Asn Val Thr Ala Pro Thr Pro Gln Gln Leu Gln Ala Phe Lys Asn 370 375 380 Glu Val Gly Val Leu Arg Lys Thr Arg His Val Asn Ile Leu Leu Phe 385 390 395 400 Met Gly Tyr Ser Thr Lys Pro Gln Leu Ala Ile Val Thr Gln Trp Cys 405 410 415 Glu Gly Ser Ser Leu Tyr His His Leu His Ile Ile Glu Thr Lys Phe 420 425 430 Glu Met Ile Lys Leu Ile Asp Ile Ala Arg Gln Thr Ala Gln Gly Met 435 440 445 Asp Tyr Leu His Ala Lys Ser Ile Ile His Arg Asp Leu Lys Ser Asn 450 455 460 Asn Ile Phe Leu His Glu Asp Leu Thr Val Lys Ile Gly Asp Phe Gly 465 470 475 480 Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His Gln Phe Glu Gln 485 490 495 Leu Ser Gly Ser Ile Leu Trp Met Ala Pro Glu Val Ile Arg Met Gln 500 505 510 Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp Val Tyr Ala Phe Gly Ile 515 520 525 Val Leu Tyr Glu Leu Met Thr Gly Gln Leu Pro Tyr Ser Asn Ile Asn 530 535 540 Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly Tyr Leu Ser Pro 545 550 555 560 Asp Leu Ser Lys Val Arg Ser Asn Cys Pro Lys Ala Met Lys Arg Leu 565 570 575 Met Ala Glu Cys Leu Lys Lys Lys Arg Asp Glu Arg Pro Leu Phe Pro 580 585 590 Gln Ile Leu Ala Ser Ile Glu Leu Leu Ala Arg Ser Leu Pro Lys Ile 595 600 605 His Arg Ser Ala Ser Glu Pro Ser Leu Asn Arg Ala Gly Phe Gln Thr 610 615 620 Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser Pro Lys Thr Pro Ile Gln 625 630 635 640 Ala Gly Gly Tyr Gly Ala Phe Pro Val His 645 650 151 base pairs nucleic acid single linear DNA (genomic) 8 AAATACAGTT ATCTGATGGT CCCTCAATTA TGTTATTTTA ATAAAATAAA TTAAATTTAG 60 GTGTAATGGC TGGCTGTTAC CTCCTTTTAA AGTAATTCTG AGCTCACAAC TTGAATGCCC 120 CATTTGTTCA CCCTCTTCAG GATCAGAATT C 151 

What is claimed is:
 1. A construct comprising a DNA segment comprising a Raf gene in an antisense orientation downstream of a promoter.
 2. The construct according to claim 1 wherein said Raf gene is Raf-1.
 3. A method of inhibiting Raf expression comprising expressing an antisense Raf gene in a cell under conditions such that said Raf expression is inhibited.
 4. The method according to claim 3, wherein said Raf gene is Raf-1.
 5. A method of inhibiting Raf kinase activity comprising: replacing a codon within the Raf gene encoding a serine or threonine amino acid for a codon encoding an amino acid not suceptable to phosphorylation and transforming said gene into a cell such that said Raf activity is inhibited.
 6. The method according to claim 5, wherein said amino acid not suceptable to phosphorylation is alanine.
 7. A method of inhibiting Raf kinase activity comprising: modifying Raf by replacing a serine or threonine amino acid within Raf for an amino acid not suceptable to phosphorylation and delivering said modified Raf to a cell such that said Raf expression is inhibited.
 8. The method according to claim 7, wherein said amino acid not suceptable to phosphorylation is alanine. 